The Sensitization And Mechanism Of CSE Inhibitor ATA On Chemotherapy Drugs In Triple Negative Breast Cancer

Objective:To study the resistance function of Cystathionine-γ-lyase（CSE）and CSE inhibitor Aurintricarboxylic acid（ATA）in triple negative breast cancer（TNBC）,and to explore the new mechanism of drug resistance in TNBC.In order to overcome the drug resistance of breast cancer,inhibit recurrence and metastasis,reduce the mortality.Methods:In the first part,the function and mechanism of CSE gene resistance in TNBC cells were studied:qRT-PCR and Western blot were used to detect CSE expression and P-gp expression in different series of breast cancer cells and normal breast epithelial cells;MTS assay was used to detect the sensitivity of TNBC cells with different CSE expression levels to cisplatin（DDP）and paclitaxel（PTX）;LV-CSE-RNAi lentivirus was used to down-regulate the level of CSE in human TNBC MDA-MB-231 cells,and LV-CSE lentivirus was used to up-regulate the level of CSE in BT549 cells.,Cell growth,proliferation,migration,invasion and apoptosis were detected by MTS,EDU,cell scratch,transwell and apoptosis assay,and the effect of CSE expression level on the sensitivity of DDP and PTX was studied.Western blot,DHE staining and reducing glutathione（GSH）kit were used to detect the effects of different CSE expression levels on SIRT1-STAT3-C-myc-Bcl-2 signaling pathway related proteins and the production of GSH and ROS in cells.In the second part,the sensitization effect of CSE inhibitor ATA on DDP and PTX in TNBC cells was studied:Cell heat transfer assay（CETSA）,Western blot and methylene blue assay were used to detect the binding ability of ATA to the target protein CSE in MDA-MB-231 cells and the effect of ATA on CSE/H₂S system;The effects of ATA combined with DDP or PTX on the growth,proliferation,migration,invasion and apoptosis of MDA-MB-231 cells were detected by MTS,EDU,cell scratch,transwell and apoptosis assay.In the third part,to study the sensitization effect of CSE inhibitor ATA on DDP and PTX in 4T1 mouse transplanted tumor model:The 4T1 mouse transplanted tumor model was constructed,and the tumor volume was about 50mm³ before intraperitoneal administration The dose of ATA was:ATA 10 mg/kg/2day,DDP 1.5 mg/kg/2day,PTX 5 mg/kg/2day,ATA plus DDP or PTX combination group,and normal saline group were used to record the changes in tumor volume and body weight of mice.The expression of Ki67 in tumor tissues was analyzed by immunohistochemistry.HE staining was used to detect the influence of drug administration on the five zang organs.In the fourth part,the mechanism of CSE inhibitor ATA sensitization to chemotherapy drugs in TNBC is explored:Western blot was used to detect the effect of ATA combined with DDP or PTX on the expression of SIRT1-STAT3-C-myc-Bcl-2 signaling pathway related proteins in triple-negative breast cancer cells and animal tumor tissues.DHE staining and GSH kit were used to detect the effects of ATA combined with DDP or PTX on the production of GSH and ROS in tumor cell microenvironment.Results:1、The results of qRT-PCR and Western blot showed that CSE was highly expressed in breast cancer cells compared with normal epithelial cells Hs578bst.2、Western blot results showed that expression of P-gp protein was positively correlated with CSE expression levels in different series of breast cancer cells.3、MTS assay results showed that the CSE-highly expressed MDA-MB-231 cells showed decreased sensitivity to DDP and PTX.4、Down-regulating CSE expression level in MDA-MB-231 cells of human TNBC cancer enhanced the inhibitory effect of DDP and PTX on the growth,proliferation,migration and invasion of MDA-MB-231cells,and enhanced the promoting effect of DDP and PTX on cell apoptosis;Up-regulation of CSE expression level in human TNBC BT549 cells reduces the inhibitory effects of DDP and PTX on the growth,proliferation,migration and invasion of BT549 cells.5、Western blot test results showed that:Down-regulation of CSE expression in MDA-MB-231 cells can promote the expression of SIRT1 protein,inhibit the expression of AC-STAT3,STAT3,P-STAT3 and the downstream Bcl-2 and C-myc protein of STAT3,thus inhibiting the SIRT1-STAT3-C-myc-Bcl-2 signaling pathway.However,the up-regulation of CSE expression in BT549 cells produced the opposite result.6、DHE staining and GSH detection results showed that:down-regulating CSE expression in MDA-MB-231 cells inhibited GSH production and promoted ROS level.The up-regulation of CSE expression in BT549 cells promoted the production of GSH and inhibited the level of ROS.7、The results of CETSA,Western blot and methylene blue experiments showed that ATA could bind to CSE and inhibit CSE/H₂S system in MDA-MB-231 cells.8、The CSE inhibitor ATA enhanced the inhibitory effect of DDP and PTX on the growth,proliferation,migration and invasion of MDA-MB-231 cells,but ATA had no significant effect on the proapoptotic effect of DDP and PTX.9、In vivo experiments showed that ATA enhanced the inhibitory effect of DDP and PTX on tumor growth in 4T1 mouse transplanted tumor model;Immunohistochemical results showed that ATA enhanced the inhibitory effect of chemotherapy drugs on Ki67 expression in tumor tissue of 4T1 mouse transplanted tumor model.HE staining showed no obvious systemic toxicity in 4T1 mouse transplanted tumor model during administration.10、Western blot results showed that:ATA combined with DDP or PTX can inhibit the CSE expression in MDA-MB-231 cells and tumor tissues,promote the expression of SIRT1 protein,inhibit the expression of AC-STAT3,STAT3,P-STAT3 and the downstream Bcl-2 and C-myc protein of STAT3.Thus,SIRT1-STAT3-C-myc-Bcl-2 signaling pathway is inhibited.11、DHE staining and GSH detection results showed that ATA can enhance DDP or PTX to inhibit GSH production in MDA-MB-231 cells,and ATA combined with DDP or PTX can promote ROS level and change the microenvironment of tumor cells.Conclusion:1、CSE is highly expressed in human breast cancer cells,and the protein expression level of CSE is positively correlated with P-gp;High expression CSE TNBC cells showed decreased sensitivity to chemotherapy drugs2、Activation of CSE/H₂S system can reduce the sensitivity of TNBC cells to chemotherapy drugs.3、The CSE inhibitor ATA can increase the sensitivity of TNBC cells to chemotherapy drugs.4、The CSE inhibitor ATA increased the sensitivity of the 4T1 mice transplantation tumor model to chemotherapy drugs,and no obvious systemic toxicity during the administration of the 4T1 mouse transplanted tumor mode.5、Change the expression level of CSE,regulate the expression of SIRT1-STAT3-C-myc-Bcl-2signaling pathway related proteins and the generation of GSH and ROS in the microenvironment of tumor cells,and participate in the influence of the sensitivity of TNBC cells to chemotherapy drugs.